Project summary. Biofilms, which are broadly defined as aggregates of cells encased by an extracellular matrix, are the predominant mode of growth for bacteria in the wild and during chronic infections. One notable property of biofilms is the production of significant levels of phenotypically diverse cells (often called phenotypic variants), which, in the opportunistic pathogen Pseudomonas aeruginosa, are the result of mutations incurred during biofilm growth. Central to the genetic and phenotypic diversification in P. aeruginosa biofilms is a novel mutagenic DSB repair pathway. Mutations that disrupt RecBC-dependent recombinational DNA repair eliminate the introduction of mutations in biofilms. But,unlike mutagenic DSB repair in Escherichia coli, mutagenic DSB repair in P. aeruginosa biofilms does not require the error-prone polymerase Pol IV, SOS response, or the stationary-phase sigma factor RpoS. The degree to which mutagenic DSB repair increases mutation frequency in P. aeruginosa biofilms is not known. The molecular mechanism by which mutations are introduced during DSB repair is also not known. The aims of this proposal are to determine and identify the mutation frequency and spectrum in P. aeruginosa biofilms, determine whether DSB repair is inherently or conditionally mutagenic in P. aeruginosa, and identify the error-prone polymerase that synthesizes DNA during DSB repair in P. aeruginosa biofilms. The research in this proposal will establish a new paradigm for mutagenic DSB repair in bacteria and lead to insights on P. aeruginosa physiology, DSB repair, and biofilms. This knowledge is crucial towards the long-term objective of understanding the process and physiological and molecular regulation of mutagenic DSB repair in P. aeruginosa biofilms, determining the biological significance of this process in chronic P. aeruginosa infections, and developing new drug targets for treatment of chronic P. aeruginosa infections. PUBLIC HEALTH RELEVANCE: Bacterial biofilms cause a significant number of chronic infections, and Pseudomonas aeruginosa is a model system for studying biofilm physiology and pathogenesis. P. aeruginosa is an opportunistic pathogen that lives in a biofilm in the lungs of patients with cystic fibrosis (CF). During chronic infections, P. aeruginosa rapidly diversifies genetically and phenotypically, adapting to the CF lung and becoming resistant to antibiotics. Knowing how biofilms generate genetic and phenotypic diversity will provide insights that apply to biofilms in general and might lead to novel treatments that inhibit mutation and adaptation of P. aeruginosa during chronic infections.